Structure and Absolute Configuration of Jurassic Polyketide-Derived Spiroborate Pigments Obtained from Microgram Quantities

Complete structural elucidation of natural products is often challenging due to structural complexity and limited availability. This is true for present-day secondary metabolites, but even more for exceptionally preserved secondary metabolites of ancient organisms that potentially provide insights into the evolutionary history of natural products. Here, we report the full structure and absolute configuration of the borolithochromes, enigmatic boron-containing pigments from a Jurassic putative red alga, from samples of less than 50 μg using microcryo­probe NMR, circular dichroism spectroscopy, and density functional theory calculations and reveal their polyketide origin. The pigments are identified as spiroborates with two pentacyclic <i>sec</i>-butyl-trihydroxy-methyl-benzo­[<i>gh</i>]­tetraphen-one ligands and less-substituted derivatives. The configuration of the <i>sec</i>-butyl group is found to be (<i>S</i>). Because the exceptional benzo­[<i>gh</i>]­tetraphene scaffold is otherwise only observed in the recently discovered polyketide clostrubin from a present-day <i>Clostridium</i> bacterium, the Jurassic borolithochromes now can be unambiguously linked to the modern polyketide, providing evidence that the fossil pigments are almost originally preserved secondary metabolites and suggesting that the pigments in fact may have been produced by an ancient bacterium. The borolithochromes differ fundamentally from previously described boronated polyketides and represent the first boronated aromatic polyketides found so far. Our results demonstrate the potential of microcryoprobe NMR in the analysis of previously little-explored secondary metabolites from ancient organisms and reveal the evolutionary significance of clostrubin-type polyketides

Resolving the Atomistic Modes of Anle138b Inhibitory Action on Peptide Oligomer Formation

The diphenyl-pyrazole compound anle138b is a known inhibitor of oligomeric aggregate formation in vitro and in vivo. Therefore, anle138b is considered a promising drug candidate to beneficially interfere with neurodegenerative processes causing devastating pathologies in humans. The atomistic details of the aggregation inhibition mechanism, however, are to date unknown since the ensemble of small nonfibrillar aggregates is structurally heterogeneous and inaccessible to direct structural characterization. Here, we set out to elucidate anle138b’s mode of action using all-atom molecular dynamics simulations on the multi-microsecond time scale. By comparing simulations of dimeric to tetrameric aggregates from fragments of four amyloidogenic proteins (Aβ, hTau40, hIAPP, and Sup35N) in the presence and absence of anle138b, we show that the compound reduces the overall number of intermolecular hydrogen bonds, disfavors the sampling of the aggregated state, and remodels the conformational distributions within the small oligomeric peptide aggregates. Most notably, anle138b preferentially interacts with the disordered structure ensemble via its pyrazole moiety, thereby effectively blocking interpeptide main chain interactions and impeding the spontaneous formation of ordered β-sheet structures, in particular those with out-of-register antiparallel β-strands. The structurally very similar compound anle234b was previously identified as inactive by in vitro experiments. Here, we show that anle234b has no significant effect on the aggregation process in terms of reducing the β-structure content. Moreover, we demonstrate that the hydrogen bonding capabilities are autoinhibited due to steric effects imposed by the molecular geometry of anle234b and thereby indirectly confirm the proposed inhibitory mechanism of anle138b. We anticipate that the prominent binding of anle138b to partially disordered and dynamical aggregate structures is a generic basis for anle138b’s ability to suppress toxic oligomer formation in a wide range of amyloidogenic peptides and proteins

Resolving the Atomistic Modes of Anle138b Inhibitory Action on Peptide Oligomer Formation

The diphenyl-pyrazole compound anle138b is a known inhibitor of oligomeric aggregate formation in vitro and in vivo. Therefore, anle138b is considered a promising drug candidate to beneficially interfere with neurodegenerative processes causing devastating pathologies in humans. The atomistic details of the aggregation inhibition mechanism, however, are to date unknown since the ensemble of small nonfibrillar aggregates is structurally heterogeneous and inaccessible to direct structural characterization. Here, we set out to elucidate anle138b’s mode of action using all-atom molecular dynamics simulations on the multi-microsecond time scale. By comparing simulations of dimeric to tetrameric aggregates from fragments of four amyloidogenic proteins (Aβ, hTau40, hIAPP, and Sup35N) in the presence and absence of anle138b, we show that the compound reduces the overall number of intermolecular hydrogen bonds, disfavors the sampling of the aggregated state, and remodels the conformational distributions within the small oligomeric peptide aggregates. Most notably, anle138b preferentially interacts with the disordered structure ensemble via its pyrazole moiety, thereby effectively blocking interpeptide main chain interactions and impeding the spontaneous formation of ordered β-sheet structures, in particular those with out-of-register antiparallel β-strands. The structurally very similar compound anle234b was previously identified as inactive by in vitro experiments. Here, we show that anle234b has no significant effect on the aggregation process in terms of reducing the β-structure content. Moreover, we demonstrate that the hydrogen bonding capabilities are autoinhibited due to steric effects imposed by the molecular geometry of anle234b and thereby indirectly confirm the proposed inhibitory mechanism of anle138b. We anticipate that the prominent binding of anle138b to partially disordered and dynamical aggregate structures is a generic basis for anle138b’s ability to suppress toxic oligomer formation in a wide range of amyloidogenic peptides and proteins

Sensitivity-Enhanced Four-Dimensional Amide–Amide Correlation NMR Experiments for Sequential Assignment of Proline-Rich Disordered Proteins

Proline is prevalent in intrinsically disordered proteins (IDPs). NMR assignment of proline-rich IDPs is a challenge due to low dispersion of chemical shifts. We propose here new sensitivity-enhanced 4D NMR experiments that correlate two pairs of amide resonances that are either consecutive (NH_<i>i</i>–1, NH_<i>i</i>) or flanking a proline at position <i>i</i>–1 (NH_<i>i</i>–2, NH_<i>i</i>). The maximum 2-fold enhancement of sensitivity is achieved by employing two coherence order-selective (COS) transfers incorporated unconventionally into the pulse sequence. Each COS transfer confers an enhancement over amplitude-modulated transfer by a factor of √2 specifically when transverse relaxation is slow. The experiments connect amide resonances over a long fragment of sequence interspersed with proline. When this method was applied to the proline-rich region of B cell adaptor protein SLP-65 (pH 6.0) and α-synuclein (pH 7.4), which contain a total of 52 and 5 prolines, respectively, 99% and 92% of their nonprolyl amide resonances have been successfully assigned, demonstrating its robustness to address the assignment problem in large proline-rich IDPs

Interdomain Dynamics Explored by Paramagnetic NMR

An ensemble-based approach is presented to explore the conformational space sampled by a multidomain protein showing moderate interdomain dynamics in terms of translational and rotational motions. The strategy was applied on a complex of calmodulin (CaM) with the IQ-recognition motif from the voltage-gated calcium channel Ca_v1.2 (IQ), which adopts three different interdomain orientations in the crystal. The N60D mutant of calmodulin was used to collect pseudocontact shifts and paramagnetically induced residual dipolar couplings for six different lanthanide ions. Then, starting from the crystal structure, pools of conformations were generated by free MD. We found the three crystal conformations in solution, but four additional MD-derived conformations had to be included into the ensemble to fulfill all the paramagnetic data and cross-validate optimally against unused paramagnetic data. Alternative approaches led to similar ensembles. Our “ensemble” approach is a simple and efficient tool to probe and describe the interdomain dynamics and represents a general method that can be used to provide a proper ensemble description of multidomain proteins

Sensitivity-Enhanced Four-Dimensional Amide–Amide Correlation NMR Experiments for Sequential Assignment of Proline-Rich Disordered Proteins

Proline is prevalent in intrinsically disordered proteins (IDPs). NMR assignment of proline-rich IDPs is a challenge due to low dispersion of chemical shifts. We propose here new sensitivity-enhanced 4D NMR experiments that correlate two pairs of amide resonances that are either consecutive (NH_<i>i</i>–1, NH_<i>i</i>) or flanking a proline at position <i>i</i>–1 (NH_<i>i</i>–2, NH_<i>i</i>). The maximum 2-fold enhancement of sensitivity is achieved by employing two coherence order-selective (COS) transfers incorporated unconventionally into the pulse sequence. Each COS transfer confers an enhancement over amplitude-modulated transfer by a factor of √2 specifically when transverse relaxation is slow. The experiments connect amide resonances over a long fragment of sequence interspersed with proline. When this method was applied to the proline-rich region of B cell adaptor protein SLP-65 (pH 6.0) and α-synuclein (pH 7.4), which contain a total of 52 and 5 prolines, respectively, 99% and 92% of their nonprolyl amide resonances have been successfully assigned, demonstrating its robustness to address the assignment problem in large proline-rich IDPs

Age-dependent circadian defects in response to wild type αS, TP-αS, EKO/Kir2.1 and NaChBac expression in DA neurons.

<p>(<b>A</b>, <b>B</b>) Double-plotted actograms of young flies (3 days after hatching) expressing wild type αS (WT-αS) (A) and TP-αS (B) under the control of the DA neuron specific <i>TH-Gal4</i> driver. T refers to circadian periodicity which is 23.8 hrs in both cases. (<b>C</b>, <b>D</b>) Double-plotted actograms of old flies (30 days after hatching) expressing WT-αS and TP-αS. Note the different circadian periodicity in response to WT-αS (T = 23.7 hrs) and TP-αS expression (T = 26.7 hrs). (<b>E</b>, <b>F</b>) Double-plotted actogram of old flies expressing EKO/Kir2.1 (E) or NaChBac (F) under the control of the DA neuron specific <i>TH-Gal4</i> driver. Note the similar extension of the circadian periodicities (T = 27.6 hrs and T = 27.0 hrs, respectively) as observed after TP-αS expression. All experiments (n = 32–58 flies) were carried out under constant dark conditions after the animals were kept in a dark-light cycle of 12∶12 hrs. T was calculated by the Chi-squared periodogram analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024701#s4" target="_blank">Materials and Methods</a>).</p

Locomotor activity and anticipation of the dark-light transition of flies expressing <i>lacZ</i>, wild type αS and the oligomer-forming TP-αS mutant.

<p>(<b>A</b>) Dark-light (D∶L = 12 hrs∶12 hrs) transition bar. (<b>B–G</b>) Locomotor activity profile of young flies (3 days after hatching; <b>B</b>, <b>D</b>, <b>F</b>) and old flies (30 days after hatching; <b>C</b>, <b>E</b>, <b>G</b>); expressing <i>lacZ</i> (blue line; <b>B</b>, <b>C</b>), WT-αS (green line; <b>D</b>, <b>E</b>) and TP-αS (red line; <b>F</b>, <b>G</b>). Black arrows point to the beginning of locomotor activity prior to the onset of light (anticipatory behavior of the flies). Note that old flies expressing TP-αS fail to anticipate the onset of the light period (red arrow in <b>F</b>). Red asterisks show the phasing out of the maximum locomotor activities after the light-dark switch. For details see text.</p

Graphene Oxide Liquid Crystals as a Versatile and Tunable Alignment Medium for the Measurement of Residual Dipolar Couplings in Organic Solvents

Residual dipolar couplings (RDCs) have proven to be an invaluable anisotropic NMR parameter for the structural elucidation of complex biopolymers and organic molecules. However, a remaining bottleneck limiting its wider use by organic and natural product chemists is the lack of a range of easily applicable aligning media for diverse organic solvents. In this study, graphene oxide (GO) liquid crystals (LCs) were developed to induce partial orientation of organic molecules to allow RDC measurements. These LCs were determined to be maintainable at very low concentrations (as low as 1 mg/mL, corresponding to quadrupolar ²H splittings ranging from 2.8 to 30 Hz and maximum ¹³C–¹H dipolar couplings of 20 Hz for camphor in a CH₃COCH₃/water system) and to be remarkably stable and broadly compatible with aqueous and organic solvents such as dimethyl sulfoxide, CH₃COCH₃, and CH₃CN. Moreover, compared with those for other alignment media, very clean and high-quality NMR spectra were acquired with the GO molecules in solution because of their rigidity and high molecular weight. The developed medium offers a versatile and robust method for RDC measurements that may routinize the RDC-based structure determination of organic molecules

Determining the Absolute Configuration of (+)-Mefloquine HCl, the Side-Effect-Reducing Enantiomer of the Antimalaria Drug Lariam

Even though the important antimalaria drug <i>rac</i>-<i>erythro</i>-mefloquine HCl has been on the market as Lariam for decades, the absolute configurations of its enantiomers have not been determined conclusively. This is needed, since the (−) enantiomer is believed to cause adverse side effects in malaria treatment resulting from binding to the adenosine receptor in the human brain. Since there are conflicting assignments based on enantioselective synthesis and anomalous X-ray diffraction, we determined the absolute configuration using a combination of NMR, optical rotatory dispersion (ORD), and circular dichroism (CD) spectroscopy together with density functional theory calculations. First, structural models of <i>erythro</i>-mefloquine HCl compatible with NMR-derived ³<i>J</i>_HH scalar couplings, ¹⁵N chemical shifts, rotational Overhauser effects, and residual dipolar couplings were constructed. Second, we calculated ORD and CD spectra of the structural models and compared the calculated data with the experimental values. The experimental results for (−)-<i>erythro</i>-mefloquine HCl matched our calculated chiroptical data for the 11<i>R</i>,12<i>S</i> model. Accordingly, we conclude that the assignment of 11<i>R</i>,12<i>S</i> to (−)-<i>erythro</i>-mefloquine HCl is correct